This is the third report for this project. The project consists of the creation of important tools for measuring T-cell receptor diversity and TCR-mediated signaling, and use of these tools in patients and experimental models of TCR diversity. The first tool is the use of high-throughput sequencing to measure the diversity of T-cell receptor rearrangements. The strategies and protocol development are ongoing and being done in collaboration with Danny Douek's lab in the NIH VRC. The sequencing method and techniques for analysis are nearly completed and patient samples will soon be analyzed using this method. The second tool is a functional measurement of diversity by stimulating patient T-cell samples with peptide libraries. This project is now very mature, as we are able to measure T-cell responses to random peptides in both the naive and memory compartments. We are now validating this method in patients with restricted repertoires. We have developed a functional screen for TCR diversity using peptides generated by random amino acids at every position except one. These 15-mer peptides provide a very diverse peptide pool, within which we believe are sufficient numbers to lead to activation of a proportion of T-cells in normal polyclonal T-cells samples. By creating individual libraries with only one fixed amino acid at a total of 3 different positions, we created a test panel of 66 peptide libraries. Each peptide library was then added to a different well of PBMC cultures of a normal volunteer, and then assayed for upregulation of CD69 and CD98 after 24 hours, or for dilution of CFSE after one week. When the panel was used on multiple normal donors, there was excellent correlation between those peptide libraries which lead to upregulation of CD69/CD98 and to CFSE dilution. The activation was dose-dependent and was blocked by MHC Class II blockade, arguing that the peptides were acting via TCR-MHC binding. We then found that this technique can also be used to prime naive T-cells by mixing sorted, naive CD4+ cells with peptide libraries loaded onto autologous DC generated from the donors monocytes. Again the stimulation was measured by CFSE dilution, could be inhibited by MHC Class II blockade, and most interestingly, could be modulated to create different Thelper phenotypes. Specifically, naive CD4 T cells could be differentiated into Th1 and Th2 cells, while low peptide doses at priming, independent of exogenous Th1 or Th2 conditions, predisposed towards Th2 differentiation, while high peptide doses led to Th1 differentiation. This system, therefore allows for the study of polyclonal human naive T cells responses to peptide stimulation and priming. We have also developed a functional, flow-based screening assay for early TCR-mediated signaling in naive Tcells, measuring calcium flux, ERK phosphorylation, and the disappearance of cytosolic IKB. Different stimuli can be used to trigger TCR signaling, including CD3 ligation, or SEB stimulation. We also are able to measure early GATA3 expression using flow cytometry within naive T cells in order to screen for cell-intrinsic predisposition to Th2 differentiation and to correlate with signaling defects when present. Using the signaling screen, we have begun to address whether impaired TCR signaling may directly explain the atopic phenotypes in patients with WAS and DOCK8 deficiency. Both are actin cytoskeleton proteins which may lead to weak TCR signaling by a number of mechanisms including direct signaling or failure to maintain proper immunologic synapses. We have found diminished ERK phosphorylation in these patients, with associated increases in early GATA3 expression within the naive T cell pool, arguing that indeed these mutations lead to impaired TCR signaling which predisposes naive T cells to produce GATA-3 early upon TCR stimulation. We have also used this screening system to identify a patient with a different T cell receptor signaling mutation. The patient was an infant with severe eczema, elevated IgE and multiple food allergies. He also had a few syndromic features such as mild pulmonic stenosis and undescended testes. ERK phosphorylation in the patient showed a rapid early peak with rapid decrease compared to control, while early GATA3 expression within his naive T cell compartment was elevated and not blocked by IL-4 blockade. Because of the pulmonic stenosis, SOS1 was sequenced, as mutations in this gene are seen in patients with Noonan syndrome which can be characterized by some of the features seen in this patient. SOS1 is a member of the Ras/MapK pathway and mutations in this pathway can cause Noonan, LEOPARD, Cardiofaciocutaneous, and Costello Syndromes-- a group of multisystem congenital disorders with significant variability, but most often associated with cardiac, neurodevelopmental and skeletal manifestations. Of interest, there are reports in the literature of allergy and immune deficiency in a subset of these patients, although they have not been well characterized, nor has the impact of this class of mutations on T cell function ever been measured. The mutations appear to lead to gain of function of the Ras/Map Kinase pathway, and mouse models have shown that many symptoms of these disorders can be cured with MEK inhibition. Lymphocyte signaling and function have not been studied in these patients, raising the question as to whether mutations in this pathway might lead to abnormal T cell differentiation and effector function. Finally, using an experimental mouse system in collaboration with the LI, NIAID and Dr Paul's lab, we have been able to show that in polyclonal responses of naive T cells primed with a specific antigen, T cells with greater affinity for the antigen tend to predominate, and lead to a Th1 phenotype. However, when these higher affinity cells are removed before priming, in this case using tetramer sorting, the resulting lower affinity T cells tend to differentiate into Th2 cells. This was further supported by cloning polyclonal T cells specific for a specific antigen and using sequence motifs of the TCR to demonstrate that lower affinity T cells tended to become Th2 clones while higher affinity T cells made less IL4.